PROJECT SUMMARY Dopamine (DA) neurons play a vital role in various brain functions including motor control, reward, emotion and memory, and thus are implicated in a number of neurological disorders, like addiction, Parkinson?s disease, schizophrenia. Accumulating evidence suggests that DA neurons also co- release other neurotransmitters in addition to DA and that such co-transmission bears behavioral outcomes, complicating the view of DAergic transmission in associated neural network. For example, recent studies have found that midbrain DA neurons have a rapid and strong inhibitory action on striatal projection neurons (SPNs). This GABAergic transmission of DA neurons behaves very similar to that of GABAergic neurons. Intriguingly, GABA release is dependent on vesicular monoamine transporter (VMAT) instead of vesicular GABA transporter, indicating that DA neurons release DA and GABA through the same pool of synaptic vesicles. However, the co-release of glutamate (Glu) from DA neurons is far more complex. First, Glu is a fast-pace electrogenic neurotransmitter whereas DA is a slow-action neuromodulator. Second, Glu is transported into vesicles in DA terminals via vesicular glutamate transporter 2 (VGluT2), which depends on a membrane potential gradient (??) instead of a pH gradient (?pH) utilized by VMAT. Third, not all axonal terminals of midbrain DA neurons release Glu. Our pilot study has suggested that VMAT-positive and VGluT2-positve vesicles are likely belongs to different populations even in the same DAergic axonal terminals and those two groups behavior differently. Therefore, we hypothesize that DA and Glu are packed and released from different synaptic vesicles in DAergic synapses with distinct kinetics and regulated by different mechanisms. To test that, we will utilize recent advances in cell reprograming technology and somatic gene modification to build an in vitro platform for sub-cellular and molecular examination of multi-neurotransmitter co-transmission in DA neurons. In particular, we will focus on (1) characterize Glu release at single synapses of DA neurons, and (2) elucidate the organization, trafficking and release kinetics of synaptic vesicles responsible for DA and/or Glu release. The outcome of this project will lead to in-depth understanding of synaptic co-transmission, a common character among numerous types of neurons in the brain. More importantly, the new methodologies and knowledge gathered in this project will pave the way for decoding the nanoscopic complexity of neurotransmission within the microscopic synapse.